Influenza has been the cause of yearly epidemics and global pandemics throughout history. The influenza virus has only eleven (11) genes, in which the multifunctional Non-Structural-1 protein (NS1) is one of 11 proteins encoded by influenza A virus (Lamb 2001; Hale et al. 2008) and is known to be indispensable for virulence. NS1 interacts with several viral and host components to subvert cellular defense mechanisms and promote viral replication via two domains: an N-terminal RNA-binding domain (RBD) and an effector domain (ED), both of which can multimerize (Hale et al. 2008). The RBD binds dsRNA, a common viral replication intermediate, thereby preventing dsRNA-dependent activation of multiple host viral defense pathways (Talon et al. 2000). The ED sequesters multiple host proteins required for cellular mRNA maturation and export and factors controlling apoptosis (Hale et al. 2008). These functions of NS1 benefit the virus by inactivating translation of new antiviral components, recruiting host translation machinery so that they can be utilized preferentially for viral mRNA translation, and extending host cell viability to ensure sufficient viral maturation can occur.
Most interactions between the influenza NS1 protein and its host targets have been identified in isolated cells grown in culture for which the assays are typically cell autonomous (e.g., viral replication, altered host gene expression). Novel targets of NS1 that might be involved in cell-cell communication were identified using Drosophila melanogaster, a well-developed genetic system for analyzing conserved signaling pathways (Reiter et al. 2001; Bier 2005) and for elucidating interactions between host and pathogen encoded proteins (Bier and Guichard 2012; Hughes et al. 2012). Due to the high degree of sequence conservation between disease genes in humans and flies, Drosophila can serve as a powerful yet inexpensive multicellular host model to study how influenza proteins interact with cellular components to manipulate the host. In contrast, most studies of viral factors have relied primarily on cell culture studies which are not well suited for identifying cell-cell interactions.
The Hedgehog (Hh) (Jiang and Hui 2008) and Notch (N) (Fortini 2009; Kopan and Ilagan 2009) signaling pathways play central roles in growth and development (Dufraine et al. 2008; Klusza and Deng 2011), tissue repair (Crosby and Waters 2010), and adult immune responses (Crompton et al. 2007; Ito et al. 2012) of vertebrates and invertebrates. In the Drosophila wing primordium (or wing disc), Hh is secreted from cells in the posterior compartment and binds to the Patched (Ptc) receptor, resulting in phosphorylation and surface accumulation of the seven pass transmembrane domain protein Smoothened (Smo) in a stripe of cells in the anterior compartment (referred to as the central organizer) (Hooper and Scott 2005). Activated Smo in turn recruits Costal-2 (Cos-2) to the plasma membrane, disrupting an inhibitory complex with the transcription factor Cubitus interruptus (Ci; Gli in mammals), thereby stabilizing and activating the full length Ci-155 protein (Wilson and Chuang 2010). In the absence of Hh signaling, microtubule associated Cos-2 promotes Ci-155 phosphorylation via cAMP-dependent Protein Kinase A (PKA) and other kinases, resulting in partial proteolysis of Ci-155 to a N-terminal repressor (Ci-75) that silences a subset of Hh target genes (Hooper and Scott 2005).
Notch signaling is required for a variety of binary cell fate decisions in many developmental contexts including specification of the dorsal-ventral compartment border in the Drosophila wing primordium (Irvine and Vogt 1997). When activated by the Delta or Serrate ligands, the Notch receptor undergoes a series of cleavage events leading to the generation of a free intracellular domain (N-ICD) (Fortini 2009; Kopan and Ilagan 2009). N-ICD forms a complex with the Suppressor of Hairless (Su(H)) protein, enters the nucleus, and acts a transcriptional cofactor to regulate Notch target gene expression.
There are very few effective drugs to treat influenza infection and these drugs (e.g., TAMIFLU) act on highly mutable extracellular proteins that function in the late phase of viral escape from the cell. Also, viral surface proteins which are the main targets for vaccines mutate rapidly to evade suppression. The fact that NS1 is involved in the pathogenicity of influenza A viruses makes it a good target to attenuate these viruses. Several studies demonstrated that influenza viruses with partial deletions in NS1 proteins are attenuated and do not cause disease, but induce a protective immune response in different species including mice (Hai et al. 2008; Talon et al., 2000), pigs (Solorzano et al, 2005; Vincent et al. 2007), horses (Quinlivan et al., 2005), birds (Steel et al., 2009) and macaques (Baskin et al., 2007). Although it had been known for more than a decade that influenza viruses with partial deletions in NS1 proteins were attenuated, all but one (Garcia-Sastre et al. 1998) NS1 truncation variants of influenza A viruses were generated by mutagenesis. Wang et al. (2008) later demonstrated that the naturally truncated variant (Garcia-Sastre et al. 1998) had propensity to generate new variants when passaged in ovo (Wang et al., 2008). The new variants were excellent live-attenuated influenza vaccine candidates (Wang et al., 2008). The ability to attenuate influenza viruses by truncation of the NS1 protein presents a great approach in design and development of the next generation live-attenuated influenza vaccines for both poultry and humans.
A significant source of mortality associated with influenza, particularly for highly virulent strains is an overly active host immune response often referred to as a cytokine storm. Cytokine storms lead to toxic-shock like symptoms and can be fatal. Agents that block cytokine storms associated with influenza or other diseases (e.g., acute allergies) could be a significant therapeutic value. Since many of the patients who secumb to highly virulent forms of influenza are young and otherwise healthy individuals, this target population may benefit most from therapies aimed at suppressing cytokine storms.
Knowledge of new host signaling responses to viral infection can lead to therapies targeted to these interactions. These treatments can be effective at suppressing the pathogenesis of many different strains of flu unlike vaccines which typically target only a few strains and require constant reformulation since the virus can evade host antibodies. There is a need to develop a new therapeutic approach for preventing and/or treating influenza through inhibiting influenza infection.